The invention relates to a system for delivering genetic material into cells in situ in a patient.
Various methods for introducing genetic material into an internal or external target site on an animal exist. Most prominent are methods of accelerated particle mediated gene transfer, such as are described in U.S. Pat. Nos. 4,945,050 (Sanford, et al.), U.S. Pat. No. 5,204,253 (Sanford, et al.) and U.S. Pat. No. 5,015,580 (Christou, et al.).
Other methods have focussed on introducing genetic material into skin cells, particularly keratinocytes. Keratinocytes are the principle cells which cover the surface of the body. They are capable of producing proteins, particularly keratin, which constitute the main surface barriers of the body. For several different reasons, keratinocytes are attractive potential targets for gene transfer. Since they are located on the surface of the body, they are easily accessed both for gene manipulation and monitoring. If complications from gene transfer would occur, for instance, the development of local tumors or local infections, these could more easily be treated in the skin than elsewhere.
To date, genetic manipulation of keratinocytes has been done in one principal way. Skin has been harvested, the keratinocytes have been separated from the fibroblasts, and then the keratinocytes individually isolated and brought into suspension. These suspensions of keratinocytes have then been cultured to confluence using tissue culture techniques, as reported by Rheinwald, J. G., Green, H. Serial Cultivation of Human Epidermal Keratinocytes: The Formation of Keratinizing Colonies From Cells. Cell G, 331-343, 1975.
The new genetic material has been introduced into the keratinocyte while being grown in vitro using either a viral vector or plasmid, as reported by Morgan, J. R., Barrandon, Y., Green, H., Mulligan, R. C. Expression of an Exogenous Growth Hormone Glue by Transplantable Human Epidermal Cells. Science, Vol. 237, 1476-1479 (1987) and Tenmer, J., Lindahl, A., Green, H. Human Growth Hormone in the Blood of Athymic Mice Grafted With Cultures of Hormone-Secreting Human Keratinocytes. FASEB J., 4:3245-3250 (1990). The transfected cells are then usually resuspended and grown on selective media in order to increase the yield of transfection. Sheets of keratinocytes are then transplanted back to the mammal from which the keratinocytes were harvested.
Even though the in vitro yield has been acceptable, the in vivo yield has been unacceptably low, both short and long term. It has been very difficult to document any significant long term (more than thirty days) expression with these techniques, for example, as reported by Garlick, J. A., Katz, A. B., Fenvjes Esitaichman, L. B. Retrovirus Mediated Transduction of Cultured Epidermal Keratinocytes. J. Invest. Dermatol., 97:824-829, 1991.
Direct gene transfer of genetic material into internal or external target sites in optional combination with the use of an xe2x80x9cin vivoxe2x80x9d culture chamber is particularly effective for long term expression of polypeptides.
Direct delivery of genetic material is accomplished by repetitive microneedle injection into intact skin cells, open skin wounds, and internal tissues or organs. In a 1 cm2 target area, microneedles make between 500 and 5000 separate injections of an aqueous solution comprising genetic material at a desired concentration.
By employing the optional culture chamber system, direct in vivo gene transfer to exposed cells in an open wound can be performed. If these cells were not covered by the chamber, they would desiccate and die. The chamber also completely seals the wound from the outside, eliminating the spread of genetic material and vectors to places outside of the wound. At the same time, the chamber prevents the accidental introduction of undesired contamination, including viruses and other microorganisms and chemical contaminants into the wound.
The use of the chamber system for gene transfer also allows non-invasive assessment of the success of transfer by assaying for the presence of the expressed protein in wound fluid, in contrast to the prior art use of invasive techniques, such as biopsies, in order to achieve the same assessment of early expression.
A wide variety of proteins and materials can be expressed, either for secretion into the general blood and lymphatic system, or to alter the properties of the protein, for example, to not express proteins eliciting an immune response against the transplanted cell.
It is an object of the present invention to provide a method for delivery of genetic material which is economical, and practical, and can be customized to the patient with minimal effort and expense.
It is another object of the present invention to provide an apparatus suitable for direct delivery of genetic material to internal target sites.